The first event in the response of target cells to insulin is the binding of the hormone to its specific receptor on the surface of cells. This binding activates an intrinsic enzymatic activity of the receptor, it catalyzes the transfer of the terminal phosphate of ATP to tyrosine residues of specific proteins. This enzymatic activity appears to be required for insulin to stimulate many of its biological responses and recently several endogenous substrates have been identified. Questions still remain however on how the tyrosine phosphorylation of these specific proteins results in subsequent biological responses. The goal of the present studies is to further elucidate this process. A better understanding of the physiologically important substrates of the insulin receptor kinase may allow one to design drugs which directly activate these proteins in cells whose receptors are impaired, a condition which may exist in non-insulin-dependent diabetics. To this end, several approaches are being pursued. First, one of the proteins that rapidly associates with the substrate called IRS-1 is itself an enzyme, a phosphatidylinositol kinase. This enzyme binds to the tyrosine phosphorylated IRS-1, its activity is stimulated and it generates two novel phospholipids, phosphatidylinositol 3,4-P and 3,4,5-P. The role of these lipids in the cell is presently not known. We have identified a protein which binds to these lipids and we propose to purify this protein, obtain a cDNA clone encoding the protein and generate antibodies to this protein to study the role of this protein in insulin signaling. In addition, we are studying another substrate of the insulin receptor kinase. By utilizing a monoclonal antibody to a 60 kDa protein we have shown that it becomes rapidly tyrosine phosphorylated in response to insulin and that it can be directly phosphorylated in vitro with the insulin receptor. This protein appears to become associated with a GTPase activating protein of ras (called GAP) but it differs from a previously observed 62 kDa tyrosine phosphorylated GAP-associated protein in SRC transformed cells. We propose to further study this protein as well to attempt to elucidate its role in mediating specific biological responses. In addition to studies of these substrates, we are attempting to develop systems for evaluating the roles of particular substrates in mediating specific responses. To this end, we have begun studies of the Drosophila insulin receptor. This receptor diverges from the human insulin receptor to a greater degree than any of the other known members of the insulin receptor family. A chimeric receptor containing the cytoplasmic domain of the Drosophila receptor with the extracellular domain of the human receptor exhibits an insulin-activated tyrosine kinase activity. Cells overexpressing this receptor will be compared to cells overexpressing the human receptor to see if one can correlate the phosphorylation of specific substrates with particular biological responses. We also plan to express this chimeric receptor and the human receptor in a line of Drosophila cells to see if any substrates are missing in this system. If so, then cDNAs encoding particular substrates can be expressed in this system to attempt to complement the receptors expressed in these cells.